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Endocrinology Vol. 143, No. 2 411-419
Copyright © 2002 by The Endocrine Society

GRH-SOMATOSTATIN-GH

A Novel Amphibian Hypothalamic Neuropeptide: Isolation, Localization, and Biological Activity

Aya Koda, Kazuyoshi Ukena, Hitoshi Teranishi, Shinji Ohta, Kazutoshi Yamamoto, Sakae Kikuyama and Kazuyoshi Tsutsui

Laboratory of Brain Science (K.U., H.T., K.T.), Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan; Core Research for Evolutional Science and Technology (K.U., H.T., K.T.), Japan Science and Technology Corporation, Tokyo 150-0002, Japan; Instrument Center for Chemical Analysis (S.O.), Hiroshima University, Higashi-Hiroshima 739-8526, Japan; and Department of Biology (A.K., K.Y., S.K.), School of Education, Waseda University, Nishiwaseda, Tokyo 169-8050, Japan

Address all correspondence and requests for reprints to: Kazuyoshi Tsutsui, Laboratory of Brain Science, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan. E-mail: tsutsui{at}hiroshima-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropeptides similar to the molluscan cardioexcitatory Phe-Met-Arg-Phe-NH2 have been identified in several vertebrates and characterized by the RFa motif at their C terminus (RFa peptides). In this study, we sought to identify an amphibian hypothalamic RFa peptide that may regulate secretion of hormones by the anterior pituitary gland. An acid extract of bullfrog hypothalami was passed through C-18 reversed-phase cartridges, and then the retained material was subjected to HPLC, initially using a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by a dot immunoblot assay employing an antiserum raised against RFa. Immunoreactive fractions were subjected to further cation exchange and reversed-phase HPLC purification. The isolated peptide was a novel RFa peptide and shown to have the sequence Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2. The cell bodies and terminals containing this peptide were localized immunohistochemically in the suprachiasmatic nucleus and median eminence, respectively. This RFa peptide stimulated, in a dose-related way, the release of GH from cultured pituitary cells, its threshold concentration ranging between 10-9 and 10-8 M. This peptide did not have any appreciable effect on the secretion of PRL and gonadotropins. It was ascertained that the peptide was also effective in elevating the circulating GH level when administered systemically. Thus, the amphibian hypothalamus was revealed to contain a novel functional RFa peptide that stimulates GH release. This peptide was designated frog GH-releasing peptide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SECRETION OF HORMONES by the anterior pituitary gland is under hypothalamic control in vertebrates. Hypothalamic neurons produce peptide factors that regulate the secretion of a particular pituitary hormone. In mammals, GH secretion from the somatotrophs is known to be under dual hypothalamic control, the primary stimulatory neurohormone being the 40- to 44-amino acid peptide GHRH, and the primary inhibitory hormone being the 14-amino acid peptide somatostatin. Although these two hypophysiotropins are thought to be the primary regulators of GH, TRH may play a role in GH regulation in some vertebrate species (1). In amphibians, GHRH (2) and primary inhibitory hormone ( 3) have been demonstrated to regulate GH secretion. Moreover, pituitary adenylate cyclase-activating polypeptide (PACAP) (2, 4) and ghrelin (5) of amphibian origin have also been shown to enhance the release of GH from the amphibian pituitary.

The molluscan neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFa) was found in the ganglia of the venus clam (6), and immunohistochemical studies using antiserum against FMRFa suggested that the vertebrate hypothalamus possesses some unknown neuropeptide similar to FMRFa. In fact, neuropeptides having the RFa motif at their C terminus have been identified in the brains of several vertebrates including mammals (7, 8, 9), birds (10, 11, 12), and fish (13). All of the identified peptides are characterized by the RFa motif at their C termini and are called RFa peptides. The striking feature of RFa peptides is that mammalian (9) and avian (11, 12) ones may regulate the release of PRL and gonadotropins, respectively. However, the exact role of RFa peptides in the regulation of pituitary hormone secretion and the mode of their actions are still uncertain in vertebrates.

In view of the immunohistochemical finding indicating that some of the FMRFa-like immunoreactive neurons project to the hypothalamic region close to the pituitary in amphibians (14, 15), we looked for an amphibian RFa peptide from bullfrog hypothalami in this study. Here we report on a novel hypothalamic peptide having the RFa motif at its C terminus, whose peptide may act on the frog anterior pituitary to stimulate GH release. This is the first hypothalamic RFa peptide with a GH-releasing activity ever reported in a vertebrate.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Peptide extraction
Adult bullfrogs (Rana catesbeiana) were used in this study. The experimental protocol was approved in accordance with the Guide for the Care and Use of Laboratory Animals prepared by Waseda University (Tokyo, Japan). Bullfrog hypothalami (n = 1000) were excised immediately after decapitation and frozen in liquid nitrogen. Frozen hypothalami were boiled for 15 min and homogenized in 5% acetic acid by using a Polytron (Kinematica, Luzerne, Switzerland) as described previously (11, 16, 17). The homogenate was centrifuged at 10,000 x g for 40 min at 4 C, and the resulting precipitate was again homogenized and centrifuged. The two supernatants were pooled and concentrated by using a rotary evaporator at 40 C. The resulting supernatant was forced through three disposable C-18 cartridges in series (Mega Bond-Elut; Varian, Harbor, CA). The retained material (RM) was eluted with 60% methanol, and the eluate was concentrated in a vacuum centrifuge.

HPLC
The concentrated material was loaded onto a C-18 reversed-phase column (10 x 250 mm; CAPCELL PAK C18 SG-120, Shiseido, Tokyo, Japan) and eluted with a linear gradient of 0–100% acetonitrile (ACN) in 0.1% trifluoroacetic acid (TFA) for 100 min at a flow rate of 1 ml/min. An aliquot of each fraction was assayed by using a dot immunoblot assay employing an antiserum raised against RFa, which immunoreagent was supplied by Dr. O. Koizumi (Fukuoka Women’s University, Fukuoka, Japan). The immunoreactive fractions were concentrated and subjected to a cation-exchange column (7.5 x 75 mm, SP-5PW, Tosoh, Tokyo, Japan) chromatography with a linear gradient of 0–1.0 M NaCl in 20 mM phosphate buffer (PB; pH 7.2) for 100 min at a flow rate of 0.5 ml/min. The immunoreactive fractions were then loaded onto another C-18 reversed-phase column (4.6 x 150 mm; ODS-80TM; Tosoh) and eluted with a linear gradient of 20–40% ACN in 0.1% TFA for 100 min at a flow rate of 0.5 ml/min.

Dot immunoblot assay
The present dot immunoblot assay was conducted according to the method described previously (18). An aliquot of each fraction was spotted onto a 0.45-µm nitrocellulose membrane (ADVANTEC MFS, Dublin, CA). The membrane was air dried at room temperature and baked for 30 min at 100 C to fix peptides on the membrane. The membrane was washed for 10 min in 0.1 M Tris buffer (pH 7.5) with 0.1% Tween 20 and 0.9% NaCl and incubated for 60 min in blocking solution containing 5% skim milk in 0.1% Tween 20 and 0.9% NaCl (blocking buffer). After blockage, the membrane was exposed for 60 min to the RFa antibody (1:1000 dilution in blocking buffer). After the primary immunoreaction, the membrane was then exposed for 60 min to antirabbit antibody linked to alkaline phosphatase (Vector Laboratories, Inc., Burlingame, CA; 1:500 dilution in blocking buffer). After washing, immunoreactive spots were detected by the reaction with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Boehringen Mannheim, Mannheim, Germany) in alkaline phosphatase substrate solution (0.5 mM MgCl2, 10 mM diethanolamine; pH 9.5; 1:50 dilution) for 20–30 min.

Structure determination
The isolated substance was subjected to amino acid sequence analysis by automated Edman degradation with a gas-phase sequencer (PPSQ-10, Shimadzu, Kyoto, Japan). Molecular weight was estimated by fast atom bombardment-mass spectrometry (FAB-MS, JMS-HX 110/110A, JEOL, Tokyo, Japan). The results of these chemical analyses suggested that the purified immunoreactive substance from bullfrog hypothalami was characterized as a novel peptide having the RFa motif at the C-terminus (frog RFa peptide). Therefore, the peptide having the suggested structure was synthesized by a manual method followed by an HF-anisole cleavage and purified by reversed-phase HPLC. Then the characterized native peptide was compared with the synthetic one with regard to the behavior on HPLC as previously described (11, 16, 17).

Antibody preparation and immunohistochemistry
Antisera were raised according to our previous method (11, 19) using the synthetic peptide linked to keyhole limpet hemocyanin with m-maleimidobenzoyl-N-hydrosuccinimide ester as the antigen. In brief, antigen solution (1 mg/ml) was mixed with Freund’s complete adjuvant (Difco, Detroit, MI) and injected sc into rabbits. After the booster injection (1 mg), blood was collected from each rabbit, and the optimum dilution of antisera was measured by the competitive ELISA described previously (11).

Immunohistochemical analysis was conducted as previously described (11, 20, 21). In brief, adult male bullfrogs were killed by decapitation (n = 6). After dissection from the skull, the brains were fixed in 4% paraformaldehyde solution overnight at 4 C and then soaked in a refrigerated sucrose solution (30% sucrose in 0.1 M PB) until they sank. Whole brains were embedded in OCT compound (Miles Inc., Elkhart, IN) and frozen-sectioned frontally or sagittally at a 20-µm thickness with a cryostat at -20 C. The section was placed on a slide precoated with 3-aminopropyltriethoxysilane (Sigma, St. Louis, MO). After blockage of nonspecific binding components with 1% normal goat serum and 1% BSA in PBS (pH 7.2) containing 0.3% Triton X-100, the sections were immersed overnight at 4 C in a 1:1000 dilution of the antiserum raised against the novel frog RFa peptide. The primary immunoreaction was followed by a 60-min incubation with rhodamine-conjugated antirabbit IgG (ICN Pharmaceuticals, Inc., Costa Mesa, CA) used at a dilution of 1:1000. The localization of immunoreactive cell bodies and fibers was examined with a fluorescence microscope (Nikon, Melville, NY). The specificity of the staining was assessed by substituting the antiserum with antiserum (1:1000 dilution) that had been preabsorbed by incubation with the antigen in a saturating concentration (20 µg synthetic frog RFa peptide/ml) overnight before use.

In vitro assay using anterior pituitary cells
Dispersed anterior pituitary cells of adult male bullfrogs were prepared as previously described (22). The completely dispersed cells were then resuspended in 67% medium 199 (M199, Nissui Pharmaceutical, Tokyo, Japan) containing 0.1% BSA. An aliquot of the cell suspension was used to count the cell number. The volume of the cell suspension was adjusted to that 1 ml contained 3 x 105 cells. Sixty thousand cells in 200 µl medium were plated in each well of a 96-multiwell plate (Corning, Inc., Corning, NY) and preincubated for 24 h at 23 C in an atmosphere at 95% air and 5% CO2. Preincubated pituitary cells were transferred to the medium containing 10-5 M frog RFa peptide and incubated for 6, 12, and 24 h at 23 C. In another series of experiments, preincubated pituitary cells were incubated in the medium containing 10-9 to 10-5 M RFa peptide for 24 h at 23 C. To confirm the responsiveness of pituitary cells, TRH (Sigma), a PRL and GH secretagogue, and mammalian GnRH (Peptide Institute, Osaka, Japan), a gonadotropin secretagogue at the concentration of 10-8 M were also used. Anterior pituitary cells were incubated with M199 alone as controls. After incubation, each medium was centrifuged, and the supernatant was subjected to RIAs for bullfrog GH (23), PRL ( 24), LH ( 25), and FSH (26).

In vivo assay using juvenile and adult frogs
Juvenile frogs weighing 12–15 g were injected ip with 40 µg RFa peptide in 100 µl saline or saline only. They were killed before and 2, 4, 8, and 12 h after the injection, and blood samples were collected. In another series of experiments, adult frogs weighing 150–200 g were anesthetized with MS 222 (Sigma), and the vena abdominalis was cannulated for injections of RFa peptide and for blood sampling according to the method described elsewhere (27). About 300 µl of blood was taken before and 1, 2, 3, and 6 h after the injection, and each time a like volume of frog Ringer solution was delivered via the cannula to compensate for the loss in total blood volume. Plasma GH concentrations were determined by RIA using the blood samples obtained in each experiment.

RIA
Highly purified bullfrog GH (28) provided by Dr. T. Kobayashi (Saitama University, Saitama, Japan), PRL (29), LH, and FSH (30) provided by Dr. S. Tanaka (Shizuoka University, Shizuoka, Japan) were radioiodinated with 125I (Na125I, Radiochemical Center, Amersham Pharmacia Biotech, Aylesbury, UK) by the method described previously (23, 24, 25, 26). The results of RIAs of bullfrog GH (23), PRL ( 24), LH (25), and FSH ( 26) assayed in duplicate aliquots were calculated in terms of ng/104 cells per 24 h. Intraassay coefficient variations in the RIA for GH, PRL, LH, and FSH were 4.1, 3.8, 3.6, and 4.3, respectively; and interassay coefficient variations, 4.3, 3.3, 3.6, and 3.9, respectively.

Statistical analysis
Results of the RIAs were expressed as the mean ± SEM. The effects of novel frog RFa peptide on the releases of GH, PRL, LH, and FSH from frog pituitaries were analyzed for significance by one-way ANOVA. If significant by ANOVA, these analyses were followed by Duncan’s multiple range test (31).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation and characterization of frog RFa peptide
An extract of hypothalami from 1000 bullfrogs was forced through disposable C-18 reversed-phase cartridges. The RM eluted with 60% methanol was then subjected to the first step of HPLC purification with a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by using a dot immunoblot assay, and immunoreactive fractions were eluted with 31–37% ACN (Fig. 1Go). These fractions were subjected to the cation-exchange HPLC, and immunoreactive fractions were eluted with 0.25–0.31 M NaCl (Fig. 2Go). Then these immunoreactive fractions were rechromatographed by the C-18 reversed-phase HPLC purification under a linear gradient of ACN (Fig. 3Go). As shown in Fig. 3Go, a substance that possessed RFa immunoreactivity appeared to be eluted as a single peak at 25% ACN. The absorbance of peak at 220 nm was 0.016.



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  		Figure 1. HPLC profile of the RM on a reversed-phase column (CAPCELL PAK C18 SG-120). The RM loaded onto the column was eluted with a linear gradient of ACN concentration (0–100%/100 min) in 0.1% TFA (pH 2.2) at a flow rate of 1 ml/min and collected in 40 fractions of 2 ml each. Aliquots (1/2000 vol) of each fraction were evaporated to dryness, dissolved in distilled water, and spotted onto a nitrocellulose membrane. The immunoreactive fractions were eluted with 31–37% ACN and are indicated by the horizontal bar.

 


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  		Figure 2. HPLC profile of immunoreactive fractions in Fig. 1Go on a cation-exchange column (SP-5PW). Elution was performed in a 100-min linear gradient of 0–1.0 M NaCl in 20 mM PB (pH 7.2) at a flow rate of 0.5 ml/min (collected in 1-ml fractions). Aliquots (1/1000 vol) of each fraction were evaporated to dryness, dissolved in distilled water, and spotted onto a nitrocellulose membrane. The immunoreactive fractions were eluted with 0.25–0.31 M NaCl and are indicated by the horizontal bar.

 


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  		Figure 3. Final purification of the immunoreactive substance by HPLC using a reversed-phase column (ODS-80TM). Elution was performed with a 100-min linear gradient of 20–40% ACN in 0.1% TFA (pH 2.2) at a flow rate of 0.5 ml/min. Aliquots (1/1000 vol) of each fraction were evaporated to dryness, dissolved in distilled water, and spotted onto a nitrocellulose membrane. The immunoreactive substance eluted with 25% ACN is indicated by the arrow.

 
Amino acid sequence analysis of the isolated substance revealed the following sequence: Ser(10)-Leu(28)-Lys(33)-Pro(28)-Ala(29)-Ala(44)-Asn(23)-Leu(13)-Pro(5)-Leu(5)-Arg(2)-Phe(2), with the detected amount (pmol) of each amino acid indicated in parentheses. FAB-MS was used to elucidate the C-terminal structure of the isolated peptide. A molecular ion peak in the spectrum of this peptide was 1326.5 m/z (M+H)+. The value indicated that the peptide was an amidated form at the C terminus.

To confirm the data obtained in the sequence analysis, we synthesized a peptide having the proposed sequence and eluted the synthetic and native peptides on two different HPLC systems (Fig. 4Go, A and B). The synthetic and native peptides showed identical retention times on the C-18 reversed-phase column (Fig. 4AGo) and on the cation-exchange column (Fig. 4BGo). The mixture of the synthetic and native peptides was also eluted as a single peak on each column (Fig. 4Go, A and B). The isolated native peptide was therefore confirmed as a 12-amino acid sequence with RFa at its C-terminus. This neuropeptide (frog RFa peptide) isolated bullfrog hypothalami has not been previously reported to exist in vertebrates.



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  		Figure 4. Comparison of HPLC behavior between native (N) and synthetic (S) peptides. A, The reversed-phase column (ODS-80TM) under an isocratic condition of 23% ACN in 0.1% TFA (pH 2.2) at a flow rate of 0.5 ml/min. B, The cation- exchange column (SP-5PW) under an isocratic condition of 0.15 M NaCl in PB (pH 7.2) at a flow rate of 0.5 ml/min.

 
Localization of frog RFa peptide in the hypothalamo-hypophysial system
The frog RFa peptide was localized in the bullfrog hypothalamus by using an antiserum raised against the peptide sequence conjugated to keyhole limpet hemocyanin. The binding of frog RFa peptide to the antibody was completely inhibited by frog RFa peptide but not by most related RFa peptides reported in other vertebrates (7, 10, 13), indicating the high specificity of the antibody (Fig. 5Go). Quail RFa peptide (11) also inhibited the binding of frog RFa peptide to the antibody because of a similar sequence (Fig. 5Go). Preadsorption of the antibody with excess frog RFa peptide resulted in the absence of immunolabeling. Therefore, this antibody was used for immunohistochemistry to elucidate the distribution of frog RFa peptide in the bullfrog brain.



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  		Figure 5. Competition of the binding of frog RFa peptide to the antiserum raised against the frog RFa peptide with various RFa peptides (molluscan RFa, see Ref. 6 ; bovine RFa, Ref. 7 ; carp RFa, Ref. 13 ; chicken RFa, Ref. 10 ; quail RFa, Ref. 11 ) by the competitive ELISA described previously (11 ).

 
As shown in Fig. 6Go, frog RFa-like immunoreactive cell bodies were localized in the telencephalon and diencephalon. No immunoreactive cells were observed in the mesencephalon or rhombencephalon (Fig. 6AGo). In the telencephalon, some immunoreactive cells were scattered around the medial septum (Sm), nucleus of the diagonal band of Broca (DB), and mediobasal telencephalon (Fig. 6Go, A and B). In the diencephalon, a few cells were present in the anterior preoptic area (POA; Fig. 6Go, A and C). The most distinct and abundant immunoreactive cells were observed in the suprachiasmatic nucleus (SCN; Fig. 6Go, A and D, and Fig. 7Go). Many immunoreactive fibers were present throughout the brain including mesencephalic and rhombencephalic regions. In particular, immunoreactive nerve endings to the median eminence (ME; Fig. 7CGo) were derived from the SCN via ventral infundibular regions (Fig. 7Go, A and B). As shown in Fig. 7Go, D–F, a complete absence of such an immunoreaction in the cell bodies and fibers was observed by preincubation of the antibody with synthetic frog RFa peptide.



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  		Figure 6. Illustrations of sagittal (A) and frontal (B–D) sections of the bullfrog brain. Immunoreactive cell bodies against the antiserum to the frog RFa peptide are shown by solid circles. The level of sections B–D is indicated by each arrow in panel A. MSA, Medial septal area; OC, optic chiasma; PD, pars distalis of the pituitary; TEL, telencephalon; OT, optic tectum; CB, cerebellum.

 


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  		Figure 7. Immunohistochemical staining of frontal (A and D) or sagittal (B, C, E, and F) brain sections of bullfrog with the antiserum to the frog RFa peptide (A–C) or with the antiserum preincubated with a saturating concentration of the peptide (D–F). PD, Pars distalis of the pituitary; PN, pars nervosa of the pituitary. Scale bars, 100 µm.

 
In vitro effect of RFa peptide on the release of GH, PRL, and gonadotropins from pituitary cells
Using primary cultures of bullfrog anterior pituitary cells, we conducted experiments to ascertain whether the frog RFa peptide localized in the hypothalamo-hypophysial system has any influence on the release of pituitary hormones such as GH, PRL, LH, and FSH. In this experiment, we first studied the effect of the frog RFa peptide (10-5 M) at varying length of incubation time (6, 12, and 24 h). As shown in Fig. 8AGo, the release of GH in the presence of the peptide was not significantly different from the control levels during the first 6 h of incubation with the peptide, but thereafter a significant increase (P < 0.01) was noted. In contrast, there was no significant effect of the peptide on the release of frog PRL (Fig. 8BGo), LH (Fig. 8CGo), or FSH (Fig. 8DGo). Secondly, we examined the effect of various concentrations of the RFa peptide on the release of GH, PRL, LH, and FSH during a 24-h incubation. As shown in Fig. 9Go, the stimulatory effect on GH release occurred in a dose-dependent manner with a threshold concentration ranging between 10-9 and 10-8 M. Again, no significant effect on the release of PRL, LH, and FSH at any concentration tested was noted (data not shown). TRH (10-8 M) employed as a standard secretagogue enhanced the release of GH and PRL up to 320% and 300%, respectively, of their control levels during a 24-h incubation. Likewise, GnRH (10-8 M) stimulated the release of LH and FSH up to 270% and 240%, respectively, of their control levels.



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  		Figure 8. Release of GH (A), PRL (B), LH (C), and FSH (D) from cultured bullfrog pituitary cells during different incubation times with (solid circles) or without (open circles) frog RFa peptide (10-5 M) at 23 C. Each circle and vertical line represent the mean of six determinations ± SEM. **, P < 0.01 (vs. control group, by Duncan’s multiple range test).

 


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  		Figure 9. Effect of frog RFa peptide on the release of GH from cultured bullfrog pituitary cells after a 24-h incubation at 23 C with different concentrations (10-9 to 10-5 M) of the peptide. Each column and vertical line represent the mean of six determinations ± SEM. **, P < 0.01; *, P < 0.05 (vs. control group, by Duncan’s multiple range test).

 
In vivo effect of RFa peptide on the circulating GH levels in juvenile and adult bullfrogs
The effect of an ip injection of RFa peptide on the GH level in juveniles became conspicuous (P < 0.01) within 2 h (Fig. 10AGo). The hormone level declined thereafter. No significant difference in the levels between the RFa peptide injected and saline-injected group was noted 4 h after the injection (Fig. 10AGo). Similarly, iv administration of the peptide to adult frogs caused a temporary rise (P < 0.01) in the circulating level of GH within 1 h, but the level determined 3 h after the injection was not significantly high, compared with the control value (Fig. 10BGo).



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  		Figure 10. Effect of frog RFa peptide on the circulating level of GH. A, Juvenile frogs received an ip injection of 40 µg RFa peptide in 100 µl saline (solid circles) or 100 µl saline (open circles). B, Adult frogs received an iv injection of 20 µg RFa peptide in 100 µl saline (solid circles) or 100 µl saline (open circles). Each circle and vertical line represent the mean of four to six determinations ± SEM. **, P < 0.01; *, P < 0.05 (vs. control group, by Duncan’s multiple range test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To identify the novel hypothalamic RFa peptide involved in the regulation of pituitary hormone secretion, we first isolated a substance possessing RFa immunoreactivity and subsequently characterized its structure in an amphibian species, the bullfrog. On the basis of the results of structure determinations, such as amino acid sequence analysis, mol wt presumption, and comparison of HPLC behavior, the isolated substance was considered to be a 12-residue peptide having the following structure: Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2 (SLKPAANLPLRFa). This isolated frog neuropeptide (frog RFa peptide) is a novel peptide.

Identification of the cells containing the isolated novel frog peptide in the brain is essential to understand its physiological role. The present immunohistochemical staining with the antibody against the isolated frog RFa peptide was restricted to cell bodies in several telencephalic and diencephalic regions. Because preadsorption of the antibody with synthetic frog RFa peptide resulted in a complete disappearance of the reaction product, the immunohistochemical staining was considered to be specific for the peptide. A striking observation in the immunohistochemical experiment was the distribution of stained cells in the SCN in the diencephalic region. The cell bodies and terminals containing this peptide were localized immunohistochemically in the SCN and the ME, respectively. It was demonstrated earlier that the frog ME receives innervation from SCN neurons (32, 33). The present immunohistochemical findings are also in agreement with these previous findings, indicating that some of the FMRFa-like immunoreactive neurons project to an area close to or within the pituitary of reptile, amphibian, or fish (14, 15, 34, 35, 36, 37, 38). Taken together, these results suggest that the isolated novel peptide acts directly on the frog anterior pituitary to regulate pituitary hormone secretion.

This hypothesis is supported, at least partly, by the results of the present in vitro bioassay with cultured anterior pituitary cells. The isolated RFa peptide stimulated, in a dose-related manner, GH release from cultured frog pituitary cells. Such a stimulatory effect can be taken as a physiological action, because its threshold concentration ranged between 10-9 and 10-8 M. We also confirmed that exogenously administered RFa peptide elevated the GH level in the circulating blood of juvenile and adult frogs. In contrast to the release of GH, this peptide did not show any appreciable effect on the basal secretion of PRL and gonadotropins from cultured frog pituitary cells. Therefore, the isolated frog RFa peptide may be a novel hypothalamic releasing factor of GH in the amphibian pituitary. To our knowledge, this is the first hypothalamic RFa peptide stimulating GH release found in a vertebrate. Thus, we designated this RFa peptide as frog GH-releasing peptide (fGRP). To date, four peptides existing in the amphibian hypothalamus, namely GHRH, PACAP, ghrelin, and TRH, have been shown to stimulate the release of GH (2, 4, 5, 39). By the present experiment, fGRP was revealed to be another hypothalamic peptide possessing GH-releasing activity. Interestingly, TRH (39, 40) and ghrelin ( 5) stimulate both GH and PRL release, whereas GHRH, PACAP (2), and fGRP (this experiment) stimulate only GH release. The existence of multiple GH-releasing peptides in the amphibian hypothalamus suggests that GH secretion in amphibians is under the multiple control in response to environmental stimuli and in relation to development, metamorphosis, and growth (41).

Recently, neuropeptides possessing the RFa motif (RFa peptide) have been found in several vertebrate brains (7, 8, 9, 10, 11, 12, 13). The frog RFa peptide isolated in the present experiment was revealed to have a high sequence homology (75%) with the quail RFa peptide (Ser-Ile-Lys-Pro-Ser-Ala-Tyr-Leu-Pro-Leu-Arg-Phe-NH2) recently isolated from the quail brain (11, 12). In addition, the C-terminal sequence of these two RFa peptides is identical with the chicken brain pentapeptide Leu-Pro-Leu-Arg-Phe-NH2 (LPLRFa), which was identified as the first RFa peptide in vertebrates (10). The possible physiological function of LPLRFa is still uncertain in higher vertebrates (10, 42, 43). One reason for this uncertainty is that there may be undiscovered members of the RFa family with different N-terminally extended forms of LPLRFa. The frog and quail RFa peptides isolated in the present and previous (11, 12) studies may be one of these forms acting as functional peptides, and chicken LPLRFa may be a fragment of chicken RFa peptide that is comparable to the frog and quail RFa peptides. In support of this view, the quail RFa peptide exerts an inhibitory effect on the release of gonadotropins from the cultured quail anterior pituitary in a dose-dependent manner with a threshold of 10-9 to 10-8 M (11). Furthermore, immunoreactivities of the quail RFa peptide were observed in the paraventricular nucleus and ME (11). Therefore, we proposed that the quail RFa peptide acts as a novel inhibitory factor of gonadotropin release in the avian hypothalamo-hypophysial system and named this peptide gonadotropin-inhibitory hormone [GnIH (11)]. In addition, another member of the RFa peptide family, found recently in the mammalian brain with a different N terminus, was shown to be a PRL-releasing peptide [PrRP (9)]. Taken together, these findings strengthen the view that there may be a group of RFa peptides contributing to the multifactorial regulation of pituitary hormone release in vertebrates.

In addition to the SCN, we found fGRP-like immunoreactive cell bodies in several telencephalic and diencephalic regions, such as the Sm, nucleus of the DB, and anterior POA. Immunoreactive fibers were also scattered throughout the frog brain including the brain stem. Judging from such a distribution pattern, this peptide might be multifunctional peptide as other RFa peptides, e.g. PrRP (44) and neuropeptide FF (45). Future studies are needed to understand the multiple regulatory functions of the RFa peptide in the frog brain.


   Acknowledgments
 
We thank Dr. Vance Trudeau (University of Ottawa, Canada) for his valuable discussion and for reading the manuscript.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan [11170237, 11354010, 12440233, 12894021, 13210101 (to K.T.), and 12440234 (to S.K.)] and by research grants from Waseda University (2000C-004), Asahi Glass Research Foundation, and Uehara Memorial Life Science Foundation (to S.K.).

Abbreviations: ACN, Acetonitrile; DB, diagonal band of Broca; fGRP, frog GH-releasing peptide; FMRFa, Phe-Met-Arg-Phe-NH2; ME, median eminence; PACAP, pituitary adenylate cyclase-activating polypeptide; PB, phosphate buffer; POA, preoptic area; RM, retained material; SCN, suprachiasmatic nucleus; Sm, medial septum; TFA, trifluoroacetic acid.

Received June 6, 2001.

Accepted for publication October 9, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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